Hematopoietic stem cells (HSC) are the cells responsible for perpetual production of blood cells in the body, and the only cells in a bone marrow transplant that provide sustained hematopoiesis. The unique property of self renewal enables HSC, in the steady state, to maintain a constant number of HSC and, in states of stress, allows them to expand their numbers by symmetric self-renewing divisions. In the first phase of this grant, we set into place a diverse set of searchers for the genes that are responsible for stem cell self-renewal. These studies revealed a strong candidate pathway involving Wnt, beta-catenin and axin; a second pathway involving Bmi-2; several other candidate genes selectively expressed in self-renewing HSC, and the unpaired/domeless-JACK-STAT pathway that specifies self-renewal in the drosophila male germ line. Here the 4 collaborating labs continue the search for the complete set of expressed genes that govern HSC behaviors including self-renewal, avoidance of apoptosis, differentiation to downstream myeloid or lymphoid lineages, and the decision to leave the bone marrow to circulate. We also introduce the use of a massive parallel sequencing (MPSS) effort to get the complete transcriptomes of LT-HSC, ST-HSC/MPP, CLP and CMP to allow electronic analyses and subtractions. We also concentrate efforts not only to continue a deep examination of the Wnt/beta?catenin signal transduction pathway (a pathway activated in oncogenesis) by quantitative methods of proteomics and system perturbation, but also the genes translocated in leukemias that have acquired the capacity to self-renew. Candidate genes identified from the library and microarray studies will be screened rapidly for function by transduction of native resting HSC with regulatable lentiviral vectors and tested in vivo and in vitro for effects on HSC numbers and functions. We continue the Drosophila genetic approach for identification of more extrinsic (hub cell) and intrinsic genes regulating spermatogenic stem cells, as well as using epistasic assays to clarify interacting gene expression networks.